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The manuscript has been improved, by adequately addressing the issues raised, and suggestions proposed by the reviewers.
Authors have revised the manuscript and fulfilled most of my suggestions.
Authors have revised the manuscript and fulfilled most of my suggestions.
Authors have revised the manuscript and fulfilled most of my suggestions.
Many methodological aspects should be addressed, including experiment replication, glycemic variability, normalization of the glycation plasma and RBC products. Also, the counterintuitive finding of blood cell glucose reduction following diet should be discussed.
The article is clearly written. Only a few typing errors should be revised. E.g. in the abstract change “inn” for “in” and remove last bracket in the last sentence of the abstract. The article is properly put into context, with enough references. Most figures are relevant and well explained, although in my opinion I would remove Figure 1 since the information that it contains is redundant with that of table 1.
The manuscript describes original research in an interesting field. The experimental design is appropriate. There are some concerns about the methodology, but the authors address it properly in the discussion. Methods are detailed, although in my opinion a brief description of the cafeteria diet used would be of interest. If metabolic cages were used to quantify ingested food it should be stated. Also, there is an allusion to unpublished results in the materials section (line 101) that is not clear what it refers to. Ethics issues have properly been addressed.
The design contemplates enough replicates and statistics have been addressed to rely on the data. I recommend adding statistics concerning the increase in body weight that is mentioned in the first paragraph of the results section. I also suggest removing lines 170-172 in which authors mention data not shown that is normalized in a way that they have just discarded as reliable enough. In the discussion section, the lines 227-229 should be better explained since the meaning is not clear. Conclusions are clearly stated.
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This manuscript is directed towards comparing differences by gender and by diet in relative glycosylation of plasma proteins, hemoglobin and red cell membrane components in rats as a means of assessing changes with an animal model of metabolic syndrome. The manuscript would come across as more focused if the background and objectives portion of the abstract contained objectives.
I have a number of concerns about this manuscript. Essentially all the data are from a single experiment with no evidence the results were reproduced. This is probably most important with the estimate of marked reduction in blood cell glucose with diet, one of the more dramatic findings. The result doesn’t make much sense physiologically, hasn’t been reproduced, and depends on a subtraction methodology full of assumptions and for which there are considerable pitfalls. The membrane glycosylation is expressed per unit phospholipid phosphorus rather than per unit protein because the authors are concerned that they can’t distinguish what protein is membrane and what protein is hemoglobin contaminant. However if that is a concern how can they depend on the fraction of the HMF generated being from membrane proteins and not from contaminating glycated hemoglobin?
The ratios of glycation residue per hemoglobin and per albumin molecule do not sound plausible. It would be well for the authors to review relevant literature and see how consistent those findings are with previous reports. It doesn’t sound like necessary positive and negative controls are reported with those determinations to support the validity of the quantitation.
The authors do not adequately explain why there are such marked dIfferences in glucoses between tail vein blood and blood obtained by exsanguination. There are some comments about effects of the anesthetic to raise blood glucose but they are phrased in a fashion that is not transparent to a reader who was not a participant in the work. An inadequate explanation of those differences tends to cast doubt on the validity of other findings obtained on those samples.
The authors allude to differences in level of glycation per unit protein between plasma proteins and RBC proteins without conveying that they understand why those are and they express surprise at the greater extent of glycation of intra-RBC proteins than of plasma proteins. There are two key concepts commonly invoked for those findings which the authors don’t convey awareness of: one is differences among animal species in the permeability of the RBC membrane to glucose and the other is the dependence of accumulation of glycated residues on the turnover time of the underlying proteins. Hb is present in the circulation for a much longer interval than albumin. There is also some evidence on correlations correlations between level of glycosylation in plasma vs in red blood cell proteins which may be of interest:
Higgins PJ, Garlick RL, Bunn HF. Glycosylated hemoglobin in human and animal red cells. Role of glucose permeability. Diabetes. 1982 Sep;31(9):743-8.
Khera PK, Joiner CH, Carruthers A, Lindsell CJ, Smith EP, Franco RS, Holmes YR, Cohen RM. Evidence for interindividual heterogeneity in the glucose gradient across the human red blood cell membrane and its relationship to hemoglobin glycation. Diabetes. 2008 Sep;57(9):2445-52.
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